Accumulation of citrullinated proteins by up-regulated peptidylarginine deiminase 2 in brains of scrapie-infected mice: a possible role in pathogenesis

Abstract

Peptidylarginine deiminases (PADs), which are a group of posttranslational modification enzymes, are involved in protein citrullination (deimination) by the conversion of peptidylarginine to peptidylcitrulline in a calcium concentration-dependent manner. Among the PADs, PAD2 is widely distributed in various tissues and is the only type that is expressed in brain. To elucidate the involvement of protein citrullination by PAD2 in the pathogenesis of brain-specific prion diseases, we examined the profiles of citrullinated proteins using the brains of scrapie-infected mice as a prion disease model. We found that, compared with controls, increased levels of citrullinated proteins of various molecular weights were detected in different brain sections of scrapie-infected mice. In support of this data, expression levels of PAD2 protein as well as its enzyme activity were significantly increased in brain sections of scrapie-infected mice, including hippocampus, brain stem, and striatum. Additionally, the expression levels of PAD2 mRNA were increased during scrapie infection. Moreover, PAD2 immunoreactivity was increased in scrapie-infected brains, with staining detected primarily in reactive astrocytes. Using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry, various citrullinated proteins were identified in the brains of scrapie-infected mice, including glial fibrillary acidic protein, myelin basic protein, enolases, and aldolases. This study suggests that accumulated citrullinated proteins and abnormal activation of PAD2 may function in the pathogenesis of prion diseases and serve as potential therapeutic targets.

Figure 1

Detection of citrullinated proteins in brains from control and ME7 scrapie-infected mice. Whole brains and various brain sections of control (lanes 1 to 3) and ME7-infected mice (lanes 4 to 6) were homogenized, and then citrullinated proteins were detected by Western blot analysis using a polyclonal anti-modified citrulline antibody. Note that citrullinated proteins were increased in scrapie-infected regions compared to controls. Each lane shows the results for a homogenate obtained from brains of each individual mouse. Asterisks indicate the citrullinated proteins. These results are representative of at least three separate experiments.

Figure 2

Immunohistochemical staining of citrullinated proteins in brain sections from control and ME7 scrapie-infected mice. Citrullinated proteins were detected in the brains of control (A–E) and scrapie-infected (F–J) mice at 160 days after inoculation. In the scrapie brain, citrullinated proteins (arrows) were more frequent than in control brains: cerebral cortex (F), hippocampus (G), striatum (H), cerebellum (I), and brain stem (J). Asterisks indicate the position of cerebellar molecular layer. Scale bar = 20 μm.

Figure 3

Comparison of expression level of PAD2 in brains from control and ME7 scrapie-infected mice. A: Expression level of PAD2 protein in whole brain and various brain sections of control and ME7-infected mice were analyzed by Western blot using a monoclonal anti-PAD2 antibody. Each lane shows the results for a homogenate obtained from dissected brain of each individual mouse. B: Densitometric analysis of bands in A normalized with β-actin. Wb, whole brain; Cc, cerebral cortex; Hi, hippocampus; St, striatum; Cb, cerebellum; Bs, brain stem. C: In whole brains, mRNA levels of PAD2 were analyzed by RT-PCR using three individuals of each group. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Expression of PAD2 in various brain sections. Immunohistochemical staining of PAD2 was performed using a monoclonal anti-PAD2 antibody in various brain sections from control (A–E) and ME7 scrapie-infected (F–J) mice. PAD2 was observed in dissected control and scrapie-infected brains at 160 days after inoculation with the ME7 scrapie strain. PAD2-positive cells were strongly immunostained in scrapie-infected brains and its immunoreactivity was observed in reactive astrocytes (arrows). A and F: Cerebral cortex; B and G: hippocampus; C and H: striatum; D and I: cerebellum; E and J: brain stem. Asterisks indicate the position of cerebellar molecular layer. Scale bar = 20 μm.

Figure 5

Cellular localization of PAD2 in scrapie-infected brains. The brain sections were doubly immunostained with anti-PAD2 and one of the following antibodies: astrocyte-specific GFAP, oligodendrocyte-specific MBP, neuron-specific NeuN, or microglia-specific B4-isolectin antibodies. Slides were examined under confocal laser-scanning microscopy. Note that PAD2-positive cells were strongly co-localized in cells positive for GFAP (arrows) with very few B4-isolectin-positive microglia (arrows). Scale bars = 20 μm.

Figure 6

Measurement of PAD2 enzyme activity in mouse brains. A: PAD2 enzyme activity in the brains of control and ME7-infected mice. PAD2 enzyme activity was determined as described in the Materials and Methods. The results are presented as a column graph of values in infected samples relative to controls. Values are mean ± SEM obtained from separate assays of three mouse brains (n = 3). B: Comparison of the expression levels of PAD2 protein in various brain sections of scrapie-infected mice. Wb, whole brain; Cc, cerebral cortex; Hi, hippocampus; St, striatum; Cb, cerebellum; Bs, brain stem. C: PAD2 enzyme activity was determined at different time points during scrapie incubation period using the whole brains of control (filled circle) and ME7-infected mice (open circle) as indicated (n = 3). D: Expression level of PAD2 protein in whole brains of control and scrapie-infected mice at 50, 100, and 150 days after inoculation was analyzed by Western blotting. Each experiment was repeated at least three times, and similar results were obtained. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Detection of citrullinated proteins from two-dimensional electrophoresis. The 2-DE gels show CBBG-250 staining of proteins from brains of control and infected mice (A and B, respectively). The rectangular areas (a–c) of the 2-DE gels represent a part of the 2-DE gels in each region of control and infected brains (left and middle, respectively) and citrullinated proteins (anti-MC) assayed by Western blot (right) from whole brain (a′), cerebellum (b′), brain stem (c′). Serial numbers in each infected region were used to distinguish between the identified citrullinated proteins in each region. The second spot in whole brain was the same as the one in brain stem, the fifth and sixth spots in whole brain were equal to the spots in cerebellum. The 19th spot was also detected in hippocampus (data not shown). Each experiment was repeated at least three times, and similar results were obtained.

Figure 8

Expression levels of several identified citrullinated proteins in brains of control and scrapie-infected mice. Proteins that had been identified as citrullinated were analyzed by Western blot in homogenates from whole brain, cerebral cortex, hippocampus, striatum, cerebellum, and brain stem. The proteins included ALDC, NSE, GFAP, MBP, and actin as a control stain. Note that ALDC and GFAP were significantly increased in scrapie-infected mice. In contrast, NSE was slightly decreased in whole brain, hippocampus, and cerebellum and MBP was significantly decreased in cerebral cortex, brain stem, hippocampus, and striatum in brains from scrapie-infected mice compared to controls.